An effective immunotherapy of established tumor requires both initial innate immune responses by NK cells and subsequent tumor-specific adaptive immune responses by T cells. IL-21 is a novel cytokine produced by activated T cells with sequence homology to IL-2 and IL-15. It regulates T, B, NK and dendritic cell functions, and plays an important role in modulating innate and adaptive immunity. Our previous studies showed that systemic administration of plasmid DNA encoding the murine IL-21 gene significantly inhibited the growth of established s.c. B16 melanoma and MCA205 fibrosarcoma in mice, and prolonged the survival of tumor-bearing mice through an NK cell-mediated mechanism. Studies from other groups have also demonstrated the in vivo antitumor activity of IL-21 through T- and NK-mediated immune responses, suggesting that IL-21 is a promising antitumor agent with therapeutic potentials in the clinic. Although IL-21 has been shown to display potent antitumor activity in vivo, its impact on adoptively transferred T cells in the immunotherapy of cancer has not been studied. In addition, it is not known whether the antitumor activity of IL-21 can be enhanced by combinational therapy with other cytokines that are known to regulate T and NK functions. Because IL-21 shares significant sequence homology and activities to IL-2 and IL-15, and can induce innate and adaptive immunity against tumor, we hypothesize that IL-21 may also enhance the antitumor activity of adoptively transferred, tumor Ag-specific T cells; and that combinational therapy with IL-21 plus other cytokines that are known to regulate NK and T-cell functions may further improve the therapeutic efficacy by enhancing innate and adaptive immune responses. The overall objective of this application is to study the immune responses and biological functions of IL-21-mediated antitumor activity, and develop new strategies for the use of IL-21 in immunotherapy of cancer. The specific aims are to: (1) determine whether IL-21, in concert with adoptively transferred, tumor Ag-specific T cells, can enhance the antitumor response in the treatment of established B16 melanoma using a newly developed pmel-1 TCR transgenic mouse model whose CD8+ T cells recognize an H-2Db-restricted epitope of the gp100(25-33) peptide, a tumor Ag widely expressed by the majority of melanomas including B16; (2) determine whether the therapeutic efficacy of combination therapy with IL-21 plus IL-15 in treatment of established tumor can be further improved through enhanced innate and adaptive immune responses.